Structure and function of the nucleolus in the spotlight
Introduction
The nucleolus is the most distinctive nuclear sub-compartment (Figure 1, Figure 2) and is the site of the biogenesis of ribosomal RNA (rRNA) and 40S and 60S ribosomal subunits (r-subunits) [1, 2, 3, 4]. In metabolically active animal and plant somatic cells and in yeast, the nucleolus contains tens to hundreds of active r-genes, which account for about one-half of the total cellular RNA production. However, most of the evidence indicates that a significant fraction, often >50%, of r-genes remains transcriptionally silent [5].
Ribosomal genes comprise a transcribed sequence and an intergenic spacer, and are located on one or (usually) several chromosomes in arrays of head-to-tail tandem repeats called nucleolus organizer regions (NORs). For instance, human diploid cells contain ∼400 r-genes on five pairs of NOR-bearing chromosomes, each NOR containing several tens of repeats. During interphase, r-repeats from more than one NOR-bearing chromosome often cluster together and participate in the formation of a given nucleolus. In mitosis, rRNA synthesis ceases as a result of phosphorylation of the relevant nucleolar factors and the nucleoli disassemble; at the end of mitosis rRNA synthesis resumes and nucleoli reform [6•, 7, 8•, 9•, 10].
Ribosome biogenesis requires RNA polymerase I (pol I)-driven synthesis, cleavage and modification of precursor rRNAs (pre-rRNAs), assembly with r-proteins and transient interactions with numerous small nucleolar ribonucleoproteins (snoRNPs) and non-ribosomal nucleolar proteins, culminating in the export of almost mature r-subunits. Although this would appear to be a simple scenario, in fact the biogenesis of ribosomes is a very much more complex process involving many more steps and factors than was foreseen [2, 11, 12••].
In the past few years, our understanding of ribosome biosynthesis has been revolutionized by proteomic research backed by genetic and biochemical analyses [2], particularly in yeast. Many of the individual steps in ribosome biogenesis have now been described at the molecular level, and most of the macromolecular components involved have been identified. However, the synthesis of these data to understand the various interactions of the different nucleolar factors and r-proteins in vivo remains at an early stage, even in yeast [2, 11, 12••, 13, 14•, 15•, 16, 17•].
Proteomic research [12••, 18, 19, 20•, 21•] is also rapidly increasing our understanding of ribosome biosynthesis in higher eukaryotes, although at a slower pace than in yeast. It is important to emphasize that, although rRNA and r-proteins have been highly conserved among eukaryotes, it is not always possible to extrapolate yeast data to metazoa and other eukaryotes. At the molecular level, for example, the factors involved in the earliest steps of the pol I activation, the pre-initiation complex (PIC), differ between yeast and mammals [4, 5]. At the cellular level, the nucleolus in the yeast Saccharomyces cerevisiae does not disassemble at mitosis, in contrast to what occurs in mammals and plants. Furthermore, important findings with relevance to human medicine are very specific to mammalian or even to human cells [22•, 23•, 24].
It has also become clear that the nucleolar localization of many factors, even ‘typical’ nucleolar components, can be altered by cell cycle, modulation of cell growth or cell differentiation and, conversely, that many ‘non-nucleolar’ proteins or macromolecular complexes are often encountered in nucleoli [3, 25]. Different location of a macromolecule suggests a different function, and indeed there is much emerging evidence that nucleoli have a number of ‘non-canonical’ functions in addition to their roles as ribosome factories, such as virus infection control, maturation of non-nucleolar RNAs or RNPs, senescence and regulation of telomerase function, regulation of cell cycle, tumor suppressor and oncogene activities, cell stress sensing and signaling [3, 7, 25, 26, 27•, 28]. The best-characterised of these other activities is in the assembly of the signal recognition particle, a complex of an RNA with several proteins that targets translation of certain proteins to the endoplasmic reticulum [25, 29, 30, 31, 32]. This activity is relatively easily rationalized as being at least connected with ribosome biogenesis and activity.
In this review, we discuss recent progress in deciphering how the processes of the ribosome biosynthetic pathway are integrated into the nucleolar structure, with the emphasis on the important conceptual changes since the last structurally oriented nucleolar review published in this journal in 1999 [33]. It is impossible to survey such a vast literature adequately in a short review, and we have had to be very selective. For the most part we have concentrated where possible on human nucleoli, but have included data from other species, particularly yeast, where relevant, or where human data is still unavailable.
Section snippets
Nucleolar constituents are enormously dynamic
Breakthrough photobleaching experiments have led to a complete reassessment of the extent of nucleolar and nuclear dynamics [7, 9•, 34, 35, 36•, 37]; similar results demonstrate the rapid diffusion of RNA [38]. Current models show that proteins freely diffuse through the nuclear space, including the nucleolus [39•], and that the mean residence time of most nucleolar proteins in nucleoli can be calculated to be only a few tens of seconds. The nucleolus exists as a discrete structure because
Pol I activity alone is not sufficient for the maintenance of nucleolar morphology
What is responsible for the maintenance of the typical steady-state nucleolar structure (Figure 1, Figure 2)? The classical explanation [33] is that pol I-driven transcription, with r-genes serving as nucleation sites, organizes and maintains nucleoli. Importantly, Gonda et al. [42] have shown in Xenopus that the maintenance of nucleolar morphology can be uncoupled from pol I-driven transcription by the action of specific intrinsic proteins. These disassembly mediator proteins, under conditions
Electron microscopy illuminates the molecular organization of active ribosomal genes
The molecular organization of active r-genes in the form of Christmas trees (CTs; Figure 1) is seen in electron micrographs of isolated and highly loosened chromatin spreads [46, 47]. We emphasize that these standard views of CTs are only obtained for certain cell types, typically yeast cells but also maturing amphibian (or insect) oocytes possessing a high number of extra-chromosomal nucleoli [48, 49]. Because of the presence of interfering chromatin structures in mammalian somatic cells, a
Where are the Christmas trees in the nucleolar forest?
EM thin-section images of eukaryotic nucleoli can be classified into three components (Figure 1, Figure 2): the fibrillar center (FC), the dense fibrillar component (DFC) and the granular component (GC). This statement, which regularly appears in publications on eukaryotic nucleoli, is extremely strong and needs to be examined critically from various structural and functional points of view.
Depending on the species, cell type and physiological state of the cell, there is considerable diversity
Conclusions
The nucleolus harbors hundreds of different biochemical processes taking place at any moment in a growing cell. This activity produces many protein and nucleoprotein complexes of different sizes, including pre-ribosomal particles. This simple fact necessarily makes any simple structural model of the nucleolus, such as the tripartite model, an oversimplification. Of course, leaving aside perinucleolar condensed chromatin masses, other nucleolar components, such as small clumps of condensed
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We apologize to the many colleagues whose work we have been unable to cite because of space limitations. We thank A Beyer, PE Gleizes, I Leger-Silvestre, Y Osheim and U Scheer for the kind gift of several micrographs, AI Lamond for allowing us to cite his unpublished data and J Mikeš for help with the preparation of the figures. Our work is funded by grants from the Ministry of Education, Youth and Sports MSM0021620806 (IR) and LC535 (IR), from the Grant Agency of the Czech Republic 304/04/0692
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